Project 3. Control of Hematopoietic Differentiation by hESCs The theoretical ability of human embryonic stem cells (hESC) to differentiate into any cell type in the body opens the possibility of improved cell based therapies. The most common cell based therapeutic approach currently used is hematopoietic stem cell (HSC) transplantation. Thus the potential for adaptation of hESC to clinical use is especially true for diseases of the hematopoietic system. To date, certain hematopoietic lineages have been derived from hESC in vitro, however the process is quite inefficient. We have found that different hESC lines vary significantly in their ability to form hematopoietic colonies in vitro, suggesting epigenetic differences in control of gene expression between these lines. Recently our laboratory also demonstrated that human T cells can be generated from hESC using a combination of in vitro co-culture of hESC on bone marrow stromal cells or embryoid body cultures, followed by introduction of partially differentiated precursors into human thymic implants in immunodeficient mice. However multi-lineage hematopoiesis in vivo has not been established, nor has the ability of hESC-derived cells to mount effective anamnestic immune responses. Together with the Cores in this Program, we propose to optimize hematopoietic differentiation, stressing T-lineage development, using both in vitro and in vivo approaches. We will investigate epigenetic control of genes relevant to T cell development, and explore the ability to reconstitute human immune responses derived from hESC in chimeric mouse models. We propose the following specific aims: 1) Optimize in vitro methods for growth and expansion of hematopoietic progenitor cells derived from hESC;2) Assess potential of hESC for T lymphoid development;3) Determine the in vivo potential for multi-lineage hematopoiesis derived from hESC. Epigenetic data will be shared with Projects 1 and 2 to build a solid database regarding control of hESC differentiation, and we will utilize novel culture surfaces produced in Core B to optimize differentiation. Thus we will leverage the resources of this Program to improve our knowledge of how to utilize hESC to reconstitute the immune system. These studies could thus be important to set the stage for hESC-based therapeutics for a wide variety of hematopoietic disorders.